In eukaryotic cells, newly made proteins are specifically transferred from their sites of synthesis to their sites of function. In most cases, this involves transport into or across at least one membrane. In general, these transport steps involve targeting elements contained within each protein's primary sequence and cellular machinery to recognize and facilitate translocation of the precursor protein. Recent studies have identified homologous protein translocation systems called "Tat" in the thylakoid membranes of chloroplasts and the cytoplasmic membranes of bacteria and archaea. Tat systems uniquely transport folded proteins using only the transmembrane proton gradient as an energy source. They do this with only three membrane components of machinery and without breaching the permeability of the membrane. The Tat system is essential in plants and some prokaryotes. Importantly, the Tat system is used by at least one human pathogen to deliver virulence factors to its hosts. As Tat components seem absent from mammalian genomes, the Tat system represents a potential target for novel antimicrobial compounds. Our long range goal is to determine the mechanism by which Tat systems translocate proteins. Our recent studies have described steps of the process and identified which of the three known components (cpTatC, Hcf106, and Tha4) participate in each step. Specifically, precursors bind to a 700 kDa receptor complex consisting cpTatC and Hcf106. Precursor binding and the proton gradient trigger assembly of Tha4 to the receptor complex. The precursor is then transported across the membrane and the translocation complex dissociates. Here we propose a series of biochemical studies into the mechanism of translocation. Specifically, purification and crosslinking studies will characterize the receptor complex composition, in situ size, signal peptide binding capabilities and binding site, and changes that it undergoes upon signal binding. A newly developed biochemical complementation assay will investigate the role of Tha4 oligomerization in translocation of folded precursors of varied size. Direct and indirect approaches will test two alternative models for the structure and operation of the translocase. The work proposed here will increase basic knowledge of the mechanisms of cellular machines. It may additionally provide for new strategies to address human microbial diseases.